PROJECT SUMMARY In order to coordinate pathogen-specific immune responses, CD4+ T helper cells must differentiate into distinct effector subtypes, including TH1, TH2, TH17, and T follicular helper (TFH) cells. T helper cell subtype differentiation is regulated at the gene expression level and occurs in response to cell-extrinsic cytokine signals. The prevailing model in the field has been that unique, lineage-defining transcription factors are activated in precursor cells by cytokine signaling and are largely responsible for establishing the cell-type specific gene expression profiles that promote T helper cell differentiation. For example, the lineage-defining transcription factor Bcl-6 has been described as as the ?master regulator? of TFH cell development. While the importance of factors like Bcl-6 is unquestioned, the notion of singular factors dictating T helper differentiation has been challenged by findings demonstrating that the differentiation of each T helper subset requires the concerted action of complex, cytokine-driven transcriptional networks, rather than the function of an individual transcription factor. The identification of the proteins that comprise these networks and the molecular mechanisms they utilize to promote cell-specific gene expression patterns will significantly advance our understanding of the regulation of T helper cell differentiation, and establish a scientific basis for the rational design of novel, increasingly effective immunotherapies. The long-term goal of our research program is to elucidate the transcriptional networks that regulate T helper cell differentiation and define the mechanisms by which subtype-specific gene expression patterns are regulated. In this application, we are focusing on the role of two notable transcription factor families: Signal Transducer and Activator of Transcription (STAT) and Ikaros Zinc Finger (IkZF) factors. Exciting preliminary data from our lab indicates that the IkZF factor Aiolos cooperates with STAT3 to induce the expression of Bcl-6. Mechanistically, a shared cytokine environment allows for increased Aiolos expression, STAT3 activation, and the formation of an Aiolos/STAT3 complex that associates with the Bcl6 promoter. Collectively, these findings provide the scientific premise for the work in this application and suggest that the interplay between these members of the STAT and IkZF transcription factor families represents a novel regulatory mechanism that promotes Bcl-6 expression and perhaps TFH cell development. Because IkZF and STAT family members are highly conserved, we propose the conceptually innovative hypothesis that the differentiation of distinct T helper subsets, including that of TFH cells, is dependent upon the formation and activity of subtype-specific IkZF/STAT complexes. We will test this hypothesis by i) elucidating the coordinated mechanisms by which Aiolos and STAT3 activate Bcl-6 expression, ii) defining the role of Aiolos/STAT3 complexes in TFH differentiation, and iii) determining whether conserved IkZF/STAT interactions regulate additional T helper cell gene programs.